Affiliation:
Departments of Physiology & Biophysics, Health Sciences Center, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794-8661 USA. wangh@pharm.stonybrook.edu.

ABSTRACT

Background: Recent advances in our understanding of cell signaling have revealed assemblies of signaling components often viewed in fluorescence microscopy as very large, irregular "punctae". These punctae are often dynamic in nature, appearing to act as mobile scaffolds that function in integrating protein-protein interactions from large arrays of signaling components. The visualization of these punctae, termed "signalsomes" when applied to protein assemblies involved in cell signaling provokes the question, what is the physical nature of these structures made visible in live cells through the expression of fluorescently-tagged fusion molecules?

Results: Steric-exclusion chromatography on wide-bore matrices, fluorescence correlation spectroscopy, and advanced proteomics permits the analysis of several important physical properties of signalsomes. Wnt canonical signaling is essential to normal cell development and dysregulation can lead to cancers in humans. Punctae/signalsomes have been reported based upon the study of fluorescently-tagged mammalian Dishevelleds. Dishevelleds are phosphoprotein scaffolds that demonstrate dynamic character and mobility in cells stimulated with Wnt3a. Recent studies have successfully isolated Dvl3-based signalsomes from mouse totipotent embryonic teratocarcinoma F9 cells in culture and sized by application of steric exclusion chromatography (SEC), displaying large discrete Mr (0.5 and 2 MDa). Activation of the Wnt canonical β-catenin/LEF-Tcf-sensitive transcriptional response leads to an upfield shift of >5 MDa of the Dvl3-based signalsome. Fluorescence correlation spectroscopy (fcs) is a single molecule analysis performed in live cells that experimentally measures the diffusion coefficient and permits calculation of MW of the signalsome (0.2 and 30 MDa species in vivo), which also reveal an upfield shift in MW in response to Wnt3a. Proteomics provides for molecular dissection of the composition of the signalsome isolated from untreated and Wnt3a-treated cells.

Conclusion: Dvl3-based punctae/signalsomes made visible by fluorescent microscopy now can be interrogated by advanced physical means, defining such properties as signalsome Mr/MW, molecular composition, and intracellular locale.

Figure 1: Steric-exclusion chromatography enables isolation and molecular sieving of large, supermolecular complexes/signalsomes. One configuration of steric-exclusion chromatography optimized in the authors' laboratory makes use of the Pharmacia AKTA-based liquid handler equipped with large diameter, long (30-60 cm) columns packed with wide-bore matrices such as Sephacryl 200, 400, or larger. The system can accommodate large sample loading and provides excellent performance in efforts designed to isolated very large supermolecular complexes like the Dishevelled3-based signalsomes highlighted in this article.

Mentions:
Based upon the size of other large supermolecular devices in cells (e.g., protein synthetic complexes [37,38], nuclear pore complex [39]), we adopted two complementary approaches [36]. The first, SEC, is capable of isolating and sizing (Mr) the complexes as well as enabling subsequent proteomic analysis, although requiring significant amounts of starting material. The second operates at the cell level using a single (or scanning) 1.5 femtoliter voxel and conditions in which expression of an autofluorescently-tagged molecule are maintained very low (5-50 molecules/cell). Under such conditions, fcs offers unparalleled analysis of calculated mass (MW) of complexes based in autofluorescently-tagged Dvl3. Isolating such "super" molecular complexes offers new challenges that render most common SEC techniques irrelevant, incapable of sieving such large molecular structures. SEC matrices more commonly found in industrial settings were exploited for these analyses. Very long column (60-90 cm), commercially packed under stringent conditions, can be adapted to provide optimal separation of large supermolecular complexes (Figure 1). Using this approach, Dvl3-based complexes, with sizes ranging from 0.2 to >5 MegaDa (MDa)-Mr became addressable for physical separation by steric-exclusion chromatography [36]. When isolated from mouse totipotent teratocarcinoma F9 cells, Dvl3-based supermolecular complex display two dominant species with Mr ~0.2 and 0.8-2.0 MDa. When Dvl3-based supermolecular complexes are isolated from F9 cells treated with Wnt3a for 30 min, the Mr of the dominant peak displays an upfield shift of >5 MDa, derived at the expense of the lower-Mr peak. The assembly of these large complexes occurs in advance of the downstream activation of the Lef/Tcf-sensitive transcription by Wnt3a. Study of Wnt canonical pathway activators versus inhibitors of signaling agree well with the ability of these treatments to stimulate versus block the assembly of Dvl3-based supermolecular complexes.

Figure 1: Steric-exclusion chromatography enables isolation and molecular sieving of large, supermolecular complexes/signalsomes. One configuration of steric-exclusion chromatography optimized in the authors' laboratory makes use of the Pharmacia AKTA-based liquid handler equipped with large diameter, long (30-60 cm) columns packed with wide-bore matrices such as Sephacryl 200, 400, or larger. The system can accommodate large sample loading and provides excellent performance in efforts designed to isolated very large supermolecular complexes like the Dishevelled3-based signalsomes highlighted in this article.

Mentions:
Based upon the size of other large supermolecular devices in cells (e.g., protein synthetic complexes [37,38], nuclear pore complex [39]), we adopted two complementary approaches [36]. The first, SEC, is capable of isolating and sizing (Mr) the complexes as well as enabling subsequent proteomic analysis, although requiring significant amounts of starting material. The second operates at the cell level using a single (or scanning) 1.5 femtoliter voxel and conditions in which expression of an autofluorescently-tagged molecule are maintained very low (5-50 molecules/cell). Under such conditions, fcs offers unparalleled analysis of calculated mass (MW) of complexes based in autofluorescently-tagged Dvl3. Isolating such "super" molecular complexes offers new challenges that render most common SEC techniques irrelevant, incapable of sieving such large molecular structures. SEC matrices more commonly found in industrial settings were exploited for these analyses. Very long column (60-90 cm), commercially packed under stringent conditions, can be adapted to provide optimal separation of large supermolecular complexes (Figure 1). Using this approach, Dvl3-based complexes, with sizes ranging from 0.2 to >5 MegaDa (MDa)-Mr became addressable for physical separation by steric-exclusion chromatography [36]. When isolated from mouse totipotent teratocarcinoma F9 cells, Dvl3-based supermolecular complex display two dominant species with Mr ~0.2 and 0.8-2.0 MDa. When Dvl3-based supermolecular complexes are isolated from F9 cells treated with Wnt3a for 30 min, the Mr of the dominant peak displays an upfield shift of >5 MDa, derived at the expense of the lower-Mr peak. The assembly of these large complexes occurs in advance of the downstream activation of the Lef/Tcf-sensitive transcription by Wnt3a. Study of Wnt canonical pathway activators versus inhibitors of signaling agree well with the ability of these treatments to stimulate versus block the assembly of Dvl3-based supermolecular complexes.

Bottom Line:
Recent studies have successfully isolated Dvl3-based signalsomes from mouse totipotent embryonic teratocarcinoma F9 cells in culture and sized by application of steric exclusion chromatography (SEC), displaying large discrete Mr (0.5 and 2 MDa).Activation of the Wnt canonical β-catenin/LEF-Tcf-sensitive transcriptional response leads to an upfield shift of >5 MDa of the Dvl3-based signalsome.Proteomics provides for molecular dissection of the composition of the signalsome isolated from untreated and Wnt3a-treated cells.

Affiliation:
Departments of Physiology & Biophysics, Health Sciences Center, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794-8661 USA. wangh@pharm.stonybrook.edu.

ABSTRACT

Background: Recent advances in our understanding of cell signaling have revealed assemblies of signaling components often viewed in fluorescence microscopy as very large, irregular "punctae". These punctae are often dynamic in nature, appearing to act as mobile scaffolds that function in integrating protein-protein interactions from large arrays of signaling components. The visualization of these punctae, termed "signalsomes" when applied to protein assemblies involved in cell signaling provokes the question, what is the physical nature of these structures made visible in live cells through the expression of fluorescently-tagged fusion molecules?

Results: Steric-exclusion chromatography on wide-bore matrices, fluorescence correlation spectroscopy, and advanced proteomics permits the analysis of several important physical properties of signalsomes. Wnt canonical signaling is essential to normal cell development and dysregulation can lead to cancers in humans. Punctae/signalsomes have been reported based upon the study of fluorescently-tagged mammalian Dishevelleds. Dishevelleds are phosphoprotein scaffolds that demonstrate dynamic character and mobility in cells stimulated with Wnt3a. Recent studies have successfully isolated Dvl3-based signalsomes from mouse totipotent embryonic teratocarcinoma F9 cells in culture and sized by application of steric exclusion chromatography (SEC), displaying large discrete Mr (0.5 and 2 MDa). Activation of the Wnt canonical β-catenin/LEF-Tcf-sensitive transcriptional response leads to an upfield shift of >5 MDa of the Dvl3-based signalsome. Fluorescence correlation spectroscopy (fcs) is a single molecule analysis performed in live cells that experimentally measures the diffusion coefficient and permits calculation of MW of the signalsome (0.2 and 30 MDa species in vivo), which also reveal an upfield shift in MW in response to Wnt3a. Proteomics provides for molecular dissection of the composition of the signalsome isolated from untreated and Wnt3a-treated cells.

Conclusion: Dvl3-based punctae/signalsomes made visible by fluorescent microscopy now can be interrogated by advanced physical means, defining such properties as signalsome Mr/MW, molecular composition, and intracellular locale.